1a.Objectives (from AD-416)
Determine the extent of variation in genetic control of freezing tolerance in wheat with the purpose of identifying wheat lines with different and new combinations of genes that confer freezing tolerance. Define the role of phospholipid-related genes in cold acclimation and freezing tolerance.

1b.Approach (from AD-416)
The overall approach is to use artificial freezing of cold-acclimated winter wheat plants, with temperature measurements taken every two minutes in the crown zone of the plants, to precisely describe the components of the freezing process that are injurious to the plants. Pharmacological agents that enhance or inhibit phospholipase enzyme activity will be used in whole-plant assays to assess their impact on cold acclimation and freezing tolerance; concomitant microarray analysis will be used to define the genes involved.

3.Progress Report
Winter wheat is exposed to a variety of freezing conditions as the seasons progress from fall to winter, but very little is known of the impact of this variation on the ability of the plants to survive into the next growing season. Twenty-five populations (5 parents and all possible progeny) were tested under rapid and gradual freezing conditions and the impact of these conditions on survival was evaluated. All populations survived to lower temperatures when frozen with gradual cooling, but by isolating the effect of temperature from the effect of chilling rate, we discovered that some populations tolerate rapid chilling better than others. This result suggested there are separate mechanisms in wheat controlling low-temperature tolerance and tolerance of rapid cooling. By genetically combining these two mechanisms, it may be possible to develop wheat lines with improved ability to survive the variable freezing conditions experienced in the field.
We previously discovered that winter wheat plants dramatically increase winter hardiness if exposed to mild freeze-thaw cycles prior to exposure to potentially damaging temperatures. Nothing is known of the genetic control of this phenomenon. We examine gene expression changes in wheat plant crown tissue in the freeze, and in the thaw, portions of freeze- thaw cycles. Over 1200 genes significantly increased expression during the freeze; nearly 100 of these were genes involved in regulation of expression of other genes. Only about 200 genes increased expression during the thaw, and very few of these were regulatory genes. These results suggested that the increased freezing tolerance brought about by exposure to mild freeze-thaw cycles largely was initiated during the freeze portion of the cycle. By identifying key genes involved in this response, and by identifying wheat lines that efficiently express these genes it should be possible to develop wheat varieties that more efficiently respond to freeze-thaw cycles and express greater winter hardiness.

4.Accomplishments
1.
Improved winter hardiness of wheat. Winter wheat is planted in the fall and must survive the winter in order to produce a crop the following summer. As autumn transitions to winter, the wheat plants are exposed to a variable range of cooling rates, warming rates, and freeze-thaw cycles. ARS scientists at Pullman, WA examined the response of wheat lines to a range of freezing and thawing conditions. Significant differences in abilities to tolerate various kinds of freezing stress were found; genetic studies indicated these differences were highly heritable. These differences can now be genetically combined and thereby improve the ability of wheat plants to survive the winter.